1. Field of the Invention
The present invention relates to chromatographs and other apparatus for separating the constituents of a sample. Particularly, the present invention relates to a miniaturized separation apparatus which comprises a porous capillary column. More specifically, the porous separation apparatus of the present invention includes a sample column and a detector that is disposed along the column to detect the presence of and identify each constituent that passes by the detector. The porous capillary column may comprise a matrix of porous silicon or hemispherical grain silicon on the surface thereof. The present invention also includes methods for manufacturing and using the inventive separation apparatus.
2. Background of Related Art
Various techniques have long been employed to separate the constituents of a sample in order to facilitate the identification and quantification of one or more of the constituents. Separation techniques are useful for separating inorganic substances and organic substances, such as chemicals, proteins, and nucleic acids. Techniques that have been conventionally employed for separating the constituents of a sample include various types of chromatography and electrophoresis.
Chromatography is a process that is employed in analytical chemistry in order to separate and identify the constituents of a sample. The various types of chromatography that have been conventionally employed include thin layer chromatography (TLC), column chromatography, gel permeation chromatography, ion-exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and gas chromatography (GC).
Thin film chromatography is a well known technique wherein a drop of a sample liquid is applied as a spot to a sheet of absorbent material, which may be paper or a sheet of plastic or glass covered with a thin layer of inert absorbent material, such as cellulose or silica gel. Thin layer chromatographic techniques typically employ a solvent mixture, such as water and an alcohol as respective stationary and mobile phases. The solvent mixture permeates the absorbent material from one edge and the capillary action of the absorbent material moves the sample across the thin layer. One of the solvents binds more tightly to the absorbent material to act as a stationary phase, while the other acts as a mobile phase. As the solvent mixture moves across the absorbent material, the constituents of the sample are separated relative to their solubility in each of the two solvents. Stated another way, the sample constituents equilibrate according to their relative solubilities in each of the solvents. Constituents which are the most soluble in the stationary phase move very little, while constituents which are more soluble in the mobile phase move at higher rates and therefore travel greater distances across the absorbent material.
Conventional column chromatography techniques employ a vertical tube, or column, that is filled with a finely divided solid, or a liquid stationary phase. As a sample is washed down through the stationary phase, it is dissolved in and carried by a mobile phase, which is typically liquid or gas. The various constituents of the sample travel through the stationary phase at different rates. Thus, each of the constituents of the sample spend a different amount of time in the column. The constituents may be collected in fractions as they exit the column and subsequently identified or otherwise analyzed. Constituents of the sample which remain in the stationary phase may be separately identified or otherwise analyzed by sectioning the stationary phase.
Gel permeation chromatography techniques typically employ a column with a stationary phase disposed therein. The stationary phase includes an absorbent gel material with pores of substantially uniform size. As the mobile phase and the sample that is dissolved therein pass through the stationary phase, some of the molecules that are smaller than the pores become entrapped therein, and therefore pass through the column more slowly. The passage of intermediately sized molecules, which are of approximately the same size as the pores, through the column is delayed some, as such molecules enter some of the pores. Molecules that are larger than the pores of the absorbent gel material pass through the stationary phase most quickly, as none of the larger molecules become entrapped in the pores.
Ion exchange chromatography is another variation of column chromatography, wherein the stationary phase comprises positively or negatively charged particles. Oppositely charged constituents of a sample are attracted to the stationary phase, and therefore pass through the column at a slower rate than uncharged constituents and constituents which have the same charge as the charged particles of the stationary phase.
In affinity chromatography, the solid phase comprises particles which have substrate molecules or particles, such as purified antibodies or purified antigens, covalently attached thereto. The substrate binds to a specific constituent or group of constituents in a sample. For example, if the stationary phase comprises antibodies that are specific for a particular antigen, as the sample and mobile phase pass through the column, only that particular antigen will be bound by the stationary phase. The remainder of the sample constituents will pass through the column quickly. The column is subsequently washed to remove any residual amount of the sample from the column. The column is then washed with a dissociating solution, such as a concentrated salt solution, an acidic solution, or a basic solution, in order to dissociate the separated sample constituent from the stationary phase.
High performance liquid chromatography (xe2x80x9cHPLCxe2x80x9d) is similar to column chromatography. In HPLC, the stationary phase is typically a liquid that is carried on very small particles, for example 0.01 mm or less. Consequently, the stationary phase has a very large surface area, and the mobile phase flows extremely slowly therethrough. Thus, a high pressure pump is typically employed in order to increase the rate at which the mobile phase moves through the column.
Conventional gas chromatography methods typically employ a liquid solid phase that is supported by a solid column and a mobile phase that comprises a substantially inert gas, such as nitrogen, argon, hydrogen, or helium. The sample is vaporized as it is injected into the column. As with thin layer chromatography, column chromatography, and HPLC, the constituents of the sample travel across the stationary phase at different rates, and therefore exit the column at different times. As the constituents of the sample exit the column, the constituents are analyzed by a detector, such as a katharometer, a flame ionizer, or an electron capture system, which generates a chromatogram. The identity of each constituent may then be determined by analyzing the chromatogram.
Gas chromatographs are ever-decreasing in size in order to increase their portability. Some small, or miniature or micro gas chromatographs, include columns, which are also referred to as capillary columns, that are fabricated on a silicon substrate. U.S. Pat. Nos. 5,583,281 (the xe2x80x9c""281 patentxe2x80x9d), which issued to Conrad M. Yu on Dec. 10, 1996; 4,935,040 (the xe2x80x9c""040 patentxe2x80x9d), which issued to Michel G. Goedert on Jun. 19, 1990; and 4,471,647 (the xe2x80x9c""647 patentxe2x80x9d), which issued to John H. Jerman et al. on Sep. 18, 1994, each disclose exemplary small silicon gas chromatography columns. The capillary columns that are disclosed in each of the ""281, ""040, and ""647 patents include open channels, or conduits, that are etched into the semiconductor substrate.
Similarly, U.S. Pat. No. 5,132,012 (the xe2x80x9c""012 patentxe2x80x9d), which issued to Junkichi Miura et al. on Jul. 21, 1992, discloses a liquid chromatograph that includes a capillary column formed in a semiconductor substrate. The capillary column of the chromatograph of the ""012 patent comprises an open channel, or conduit.
U.S. Pat. No. 5,571,410 (the xe2x80x9c""410 patentxe2x80x9d), which issued to Sally A. Swedberg et al. on Nov. 5, 1996, discloses a miniature gas chromatography system which includes a capillary column that is formed in a non-silicon substrate by laser ablation. The capillary column of the chromatograph of the ""410 patent comprises an open channel, or conduit, with a substantially smooth surface.
The use of substantially smooth, open-channeled capillary columns in miniature chromatographs is, however, somewhat undesirable from the standpoint that open-channeled columns typically have a surface area that is limited by the area of the substantially smooth surface of the channel. The amount of stationary phase material that may be disposed along a given length of substantially smooth, open-channeled capillary columns is also limited by the surface area of that length of the capillary column. Thus, in order to effectively separate the various constituents of a sample, the capillary column must be relatively long. Consequently, the substrate on which the capillary column is formed must have a sufficient surface area to facilitate fabricating the capillary column thereon. Thus, the use of substantially smooth, open-channeled capillary columns in miniature gas chromatographs imposes minimum size limitations on such chromatographs.
Another technique for separating the various constituents of a sample is typically referred to as electrophoresis. Electrophoresis is a process whereby molecules having a net overall electrical charge are migrated at a rate that depends on the electrical charge, size and shape of the molecule. Electrophoresis techniques typically employ a solid matrix through which the constituents, or molecules, of the sample are migrated. A variation of electrophoresis that is typically referred to as polyacrylamide gel electrophoresis (PAGE) separates molecules based strictly on their size. In PAGE, the molecules of the sample are typically linearized and separated, or disassociated from themselves and from other molecules, by means of sodium dodecyl sulfate (SDS), a detergent that binds to the hydrophobic regions of proteins, and 2-mercaptoethanol, or xcex2-mercaptoethanol, which breaks disulfide (Sxe2x80x94S) linkages that occur between some amino acids of a protein. The sample is then migrated through a polyacrylamide gel cross-linked matrix, which has very small pores. The pore size of the polyacrylamide gel may be adjusted in accordance with the molecular size, or weight, range for which separation is desired.
The preparation of polyacrylamide gels is a relatively long process. Moreover, the acrylamide that is used to form the gel matrix is a neurotoxin. Some of the other chemicals that may be utilized in electrophoretic processes are also hazardous. In addition, the amount of electric current that may be used to separate the constituents of a sample in gel electrophoresis has conventionally been limited, as too great a current will melt or otherwise disrupt the structure of the gel.
Thus, a small separation apparatus is needed that may be employed to conduct various types of sample separation, which is smaller than conventional devices, and which separates samples adequately. There are also needs for reduced equipment and operational costs.
The separation apparatus, method of manufacturing the separation apparatus, and methods of using the separation apparatus of the present invention address each of the foregoing needs.
The sample separation apparatus of the present invention includes a substrate with a capillary column thereon, the latter comprising a rough surface, such as a matrix which defines a plurality of pores therethrough or an open column with a rough surface, which is also referred to as a matrix. The surface area of the matrix of each capillary column facilitates the separation of the constituents of a sample over a relatively short length of the column compared to the required lengths of conventional smooth, xe2x80x9copenxe2x80x9d, etched or ablated columns to effectively separate the constituents. Preferably, the capillary column, which is also referred to as a porous capillary column, comprises porous silicon or hemispherical grain silicon, and is formed on a silicon substrate. Such a column, depending on the width and depth thereof, may be useful for separating the constituents of a sample or detecting constituents in a sample having a volume of as small as about one femtoliter (1xc3x9710xe2x88x9215 liter). The separation apparatus may also include a detector disposed proximate the capillary column. Such a detector analyzes a characteristic of a constituent as the constituent passes through the capillary column, and thereby identifies or otherwise analyzes the constituent.
In a first variation of the apparatus of the present invention, the sample separation apparatus may be employed as a chromatography column. Accordingly, a stationary, or solid, phase is disposed on the matrix of the capillary column. The type of stationary phase that is selected for use in the sample separation apparatus is dependent upon several factors, including without limitation the chromatographic technique that will be employed with the separation apparatus and the type of sample constituents that are to be isolated. The types of stationary phase materials that are useful in conventional chromatographic processes are also useful in the first variation of the separation apparatus.
A second variation of the separation apparatus of the present invention is useful for conducting electrophoretic separation. Thus, size of the pores that are defined through the porous silicon matrix or the amount of space between grains of hemispherical grain silicon of the capillary column is determined by the desirable rate of separation and the size of the sample constituents for which separation is desired. The second variation of the separation apparatus also includes first and second electrodes positioned proximate respective first and second ends of the capillary column. The first and second electrodes are connectable to opposite electrical charges so as to facilitate the generation of a current along a length of the capillary column, and thereby facilitate the movement and separation of the sample constituents along the column. Preferably, the second variation of the separation apparatus also includes a control column adjacent the capillary column and having substantially the same dimensions, structure, and pore sizes or spacing as the capillary column. The control column is useful for determining the molecular size or weight of at least some of the various sample constituents.
In a third variation of the apparatus, the sample separation apparatus may be employed to detect the presence or absence of increased levels of a certain analyte. Accordingly, the third variation includes a capture substrate disposed on at least a portion of the rough surfaces of the capillary column. Preferably, the capture substrate has a specific affinity for the measured, or assayed, analyte.
A method of fabricating the sample separation apparatus of the present invention includes selectively forming a capillary column in a substrate.
When a silicon substrate is employed, various techniques which are known in the art may be employed to define a porous silicon capillary column therein. Known techniques may also be used in order to form pores of a desired size. Known semiconductor layer formation processes may also be employed to fabricate a detector proximate the capillary column. Similarly, known processes are useful for fabricating electrodes and other structures upon a surface of the substrate.
Capillary columns that include hemispherical grain silicon may also be selectively formed in a substrate by known techniques. First, a trench, which defines the path of the capillary column, is defined in a substrate by known patterning processes, such as mask and etch techniques. The surface area of the surfaces of the trench may then be increased by known methods, such as by forming hemispherical grain silicon thereon.
A method of utilizing the inventive separation apparatus includes disposing a sample proximate an end of the porous capillary column and drawing the sample through the porous capillary column to generate a flowfront of the sample and effect the separation of a constituent from the sample. The sample may be drawn along the capillary column by positive pressure, negative pressure, capillary action, electric current, or any other known technique that is employed to facilitate the movement of a sample along a separation apparatus.
Variations of the inventive method employ the separation apparatus of the present invention to effect various separation techniques, including, without limitation, various types of chromatographic separation, electrophoresis, and the isolation and detection of one or more analytes from a sample.